【作者】 刘玮琦；

【导师】 谢丙炎；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2008， 硕士

【摘要】 随着我国保护地栽培面积的扩大,保护地栽培措施的一些弊端日益显现出来,其多年连作、过量施用化肥、高温高湿、蒸发量大等特点,形成了有悖于露地土壤的物理、化学和生物学的特性,从而造成了土壤板结酸化、土壤养分失衡、土壤次生盐渍化、土传病害的严重发生、土壤微生物区系失衡、土壤根结线虫为害严重等各种问题。土壤微生物在土壤生态系统中具有不可替代的重要作用,它们的群落组成失衡将直接破坏土壤中的生态平衡,对土壤环境和农业生产带来严重影响。因此本研究应用16S rRNA基因克隆文库技术研究在保护地栽培条件下,土壤微生物群落区系结构的组成以及可能发生的变化,对于弄清保护地土壤微生物的群落多样性的变化给土壤和植物带来的影响提供理论依据,并对于保护地农业的可持续发展等问题具有重要意义。氮是植物生长所必需的营养元素,是土壤生产力的重要限制因素。土壤中氮循环的不平衡将影响到其他重要的生物地球化学循环,乃至全球环境的变化。保护地栽培环境改变了其覆盖地区土壤的生态环境,高温、高湿、高肥以及CO2浓度增高等都直接影响土壤中与氮素循环密切相关的氨氧化菌的群落结构组成。因而通过对保护地和露地土壤氨氧化菌amoA基因克隆文库的构建,研究保护地栽培条件下可能对土壤氨氧化菌群落多样性产生的影响,对于进一步研究农田氮素循环和全球环境变化等重大问题具有重要的意义。本研究主要结果如下:1、通过保护地(根结线虫发生地)和露地(健康)菜田土壤细菌(Bacteria)和古菌(Archaea)16S rRNA基因克隆文库的构建,对两地土壤细菌和古菌群落多样性的系统发育分析表明:(1)应用ARDRA技术对两地土壤细菌(Bacteria)和古菌(Archaea)多样性分析表明,露地土壤细菌和古菌的群落组成多样性均高于保护地栽培条件下两种群的多样性。(2)利用∫-Libshuff等生物信息技术等对两地土壤细菌(Bacteria)和古菌(Archaea)16S rRNA基因克隆文库的系统发育和比较分析表明:两地土壤细菌(Bacteria)群落区系结构组成没有显著的差异,但保护地根结线虫地土壤农杆菌(Agrobacterium)所占的比例较大,且其中能引起植物根癌病的Agrobacterium tumefaciens数量很大。保护地土壤农杆菌(Agrobacterium)的克隆数目是露地土壤农杆菌数目的3.66倍;利用∫-Libshuff计算两地土壤古菌(Archaea)克隆文库的P值均为0.0000(P<0.05),因此得到两古菌(Archaea)克隆文库存在显著不同,其中Euryarchaeota在保护地土壤和露地土壤古菌16S r RNA克隆文库中比例相差非常大,分别为19.0%和1.92%。(3)两地土壤细菌和古菌多样性分析表明,露地土壤中的多样性组成均高于保护地土壤。土壤的微生物种类不同其功能也很不同,因此微生物多样性的降低,将直接影响到各种微生物不同功能的发挥,以及相互间的互作。从而对土壤各种营养元素的循环、有机物的分解利用,植物对养分的吸收和生长发育产生严重的不良影响,破坏植物正常的生长环境从而使各种植物病害普遍发生。2、通过保护地和露地土壤细菌(Bacteria)和古菌(Archaea)amoA基因克隆文库的构建,对两地土壤氨氧化古菌(AOA)和氨氧化细菌(AOB)群落多样性的系统发育分析表明:(1)利用DOTUR软件对克隆文库丰富度(richness)和Chao1评估的丰度(Chao1-estimated richness)参数的比较分析表明:露地菜田土壤中的氨氧化菌(AOA和AOB)的种群多样性均明显高于保护地栽培条件下氨氧化菌的种群多样性。(2)利用∫-Libshuff等生物信息技术对两地氨氧化菌(AOA和AOB)amoA基因克隆文库的系统发育分析和比较分析表明:两地土壤氨氧化古菌(AOA)克隆文库的P值均为0.0000(P<0.05)。则得到两克隆文库显著不同;同时保护地土壤氨氧化细菌克隆文库与露地菜田氨氧化细菌(AOB)克隆文库的P值也均为0.0000(P<0.05),两克隆文库也存在显著的不同。因此得出保护地环境条件下土壤微生物中与氮循环有关的氨氧化菌的群落组成情况发生了显著的变化。(3)露地土壤中氨氧化菌的多样性组成均显著高于保护地土壤。本研究推测在保护地这一环境条件下由于氨氧化菌的多样性的降低,很可能使硝化作用变得脆弱和敏感,并且很容易受到干扰,硝化作用的效率降低,对复杂环境的适应能力显著减弱,进而严重影响土壤氮素循环的正常运转,从而进一步对土壤自身的养分循环、土壤生态系统的平衡、植物的生长发育带来诸多不良影响。更多还原

【Abstract】 With the expansion protected area in China, the shortcomings of the protection cultivation measures gradually emerged.cultivation for many years, excessive use of fertilizers, high temperature and humidity, evaporation and other major features, soil formation contrary to the physical, chemical and biological properties. Resulting in the acidification of soil compaction, soil nutrient imbalance, soil salinization, soil-borne diseases a serious outbreak, soil microbial flora imbalance in the soil root-knot nematode damage and other serious problems. Soil Microbial in the soil ecosystems have an irreplaceable role .So that soil Microbial imbalance in will directly undermine the biological balance, product a serious impact on soil environment and agricultural production.In this study, Application of 16S rRNA gene cloning library technology research in the protection of cultural facilities to study the district of soil microbial community structure and change of the composition. To provide a theoretical basis for the diversity of soil microorganisms in the soil and the impact to the plants. There is great significance for the sustainable the development of protection agriculture and other issues.Nitrogen is essential nutrition element for plant growth, and a major limiting factor for soil productivity. Soil nitrogen cycle imbalance will affect other important biogeochemical cycles, and global environmental change. Protection of the cultural facilities changed the ecological environment of its coverage of the soil, high temperatures, high humidity, high-fat and higher CO2 concentration, that will directly affect the community structure of the ammonia-oxidizing bacteria and Archaea which are closely related to the nitrogen cycle. Therefore application of ammonia-oxidizing Bacteria and Archaea amoA gene library research the impact of the structure and the composition of soil microbial community in the protection of cultural facilities under the conditions of soil root knot nematode disease occurred areas, for further study nitrogen cycle of farmland and global environmental change is of great significance. The main results were as follows:1.The soil Bacteria and Archaea amoA 16S rRNA gene libraries were generated for the soil of protected cultivation and open field, the diversity and phylogenetic analysis of Bacteria and Archaea in the two soil showed that:(1) Application ARDRA technology on both the soil bacteria and Archaea diversity analysis showed that the bacteria and archaea community composition and diversity in the open field with health of soil are higher than the protection of cultural facilities root knot nematode took place.(2) These observed variations in community composition were statistically compared using∫- Libshuff. The Phylogenetic analysis and comparative analysis showed that: the department of community composed of two soil bacteria is no significant differences, but in the protection of cultural facilities root knot nematode took place,soil Agrobacterium has a larger proportion than the open field . Agrobacterium tumefaciens which can cause cancer of plant has the large numbers. the number of clones of soil Agrobacterium is 3.66 times than the open field; The P values of the two Archaea cloning library are 0.0000 (P <0.05), that calculate with the∫-Libshuff. So that the two Archaea clone libraries exists significant differences between them .The number of clones of the Euryarchaeota in the tow libraries of Archaea 16S rRNA gene are very difference, respectively 19.0 percent and 1.92 percent.(3) Bacteria and archaea diversity of the two soil analysis showed that the soil in the open field were higher than the protection land of biodiversity components. Different types of soil micro-organisms are very different functions, so the reduction of microbial diversity, will directly affect the different functions of the various micro-organisms, and the interaction between themselves. Thus the various soil nutrient cycle, the decomposition of organic matter, plant nutrient absorption and the growth and development will have serious adverse effects.2.The soil Bacteria and Archaea amoA gene library were generated for the soil of protected cultivation and open field, the two soil ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) diversity of the phylogenetic analysis showed that:(1) To assess AOA and AOB diversity within the soil of protected cultivation and open field, the OTUs predicted by richness and Chao1-estimated richness. the analysis showed that: the diversity of ammonia-oxidizing bacteria (AOA and AOB) in the open vegetable fields are significantly higher than the soil of protected cultivation.(2) These observed variations in community composition were statistically compared using∫- Libshuff. The phylogenetic analysis and comparative analysis of the libraries showed that: the P value of two ammonia-oxidizing archaea (AOA) clone libraries are 0.0000 (P <0.05). there is significant differences between the two libraries. the P value of two ammonia-oxidizing bacteria (AOB) clone libraries are 0.0000(P<0.05). two clone libraries also significantly different. All this show that the composition of the community of the AOA and AOB that related to the nitrogen cycle are significant change.(3) the diversity of ammonia-oxidizing bacteria and archaea were significantly higher than that of the protection land. So speculated that because of ammonia-oxidizing bacteria and archaea the diversity of the lower under the conditions of the protected land, the nitrification is likely to become fragile and sensitive. And are susceptible to interference, reduced the efficiency of nitrification, weakened the ability to adapt to the complex environment significantly. Then serious impact nitrogen cycle of normal operation in soil. Thus further to adverse affect soil nutrient cycling and soil ecosystem balance, plant growth and development.更多还原